Divergence in gene regulatory networks plays a major role in the evolution of every kingdom of life. While comparative studies of system evolution have been remarkably effective in identifying the functional genomics elements and tracing major evolutionary events, most studies have relied on sequence data alone. There have been few studies done in yeasts combining sequence and functional genomic data, and they underscore the power of comparative functional genomics in analyzing the function and evolution of molecular systems. Early results have been promising, but are limited in phylogenetic or biological scope, and have had no choice but to employ ad hoc approaches to the study of system evolution. This proposal aims to systematically analyze the structure and evolution of the gene regulatory network underlying a conserved stress response in a tractable subset of Ascomycota fungi. The species in this study span more than 300 million years of evolution and include the model organisms S. cerevisiae and S. pombe, as well as the human fungal pathogens C. albicans and C. glabrata. The gene regulatory network is controlled by Hog1, a highly conserved MAP-Kinase (p38 in humans) which in budding yeast evokes a stress response to facilitate survival in challenging osmotic conditions and mediates general stress response in fission yeast and fungal human pathogens. By analyzing the transcriptional program of the Hog1 stress response in a strategic subset of six species of Ascomycota, this work will distinguish the conserved, essential components of the response from derived components. To accomplish this goal, three aims will be pursued: 1) Identify genes in the Hog1-activated Osmotic Stress Response (OSR) in each of six Ascomycota species by measuring global gene expression differences between wild-type and strains lacking Hog1;2) Map the cis-regulatory elements of the Hog1- activated OSR genes in each of the six species;and 3) Construct quantitative models of the Hog1-activated OSR network in three of the six species, thereby adding depth to complement the breadth of the study. Accomplishing these aims will enable the reconstruction of the ancestral Hog1 gene regulatory network from six highly informative clades of Ascomycota fungi. Species- and clade-specific components will provide insights into lifestyle requirements and inform understanding of the ecologies and/or histories of those species. Additionally highly conserved, or convergent components, perhaps such as those specific to pathogens, may generate new therapeutic targets. Finally, understanding gained from systematically identifying gene regulatory changes across broad evolutionary space will provide significant insight into the forces shaping gene regulatory programs - forces with implications in all human disease and especially cancer.